Affinity filters, a new approach to the isolation of tox mutants of Vibrio cholerae

Abstract

We have devised a novel plate assay method for detecting mutants of Vibrio cholerae altered in the production of cholera toxin (tox mutants). Colonies replicated from a master plate are grown on the surface of a cellulose filter disc to which ganglioside-albumin conjugates have been attached. Toxin secreted by the colonies is tightly bound to the ganglioside filters. After removal of the cells by washing, the bound toxin may be detected by treating the filters with radioactively labeled antibodies against either whole toxin or one of its constituent polypeptide chains, followed by autoradiography. Colonies producing significantly greater of lesser amounts of toxin than the parental type are easily recognized and can be shown in liquid culture to have the corresponding hypertoxinogenic or hypotoxinogenic phenotype. This method, termed "the ganglioside filter assay," is applicable to screening large numbers of colonies and should facilitate isolation of various specific classes of mutants in cholera toxin production. In modified form the method will be applicable to various systems in which mutants of secreted proteins are sought.